Abstract:

The invention relates to a nutraceutical composition such as infant
formula or infant food comprising a) a defined aroma composition; b) a
methodology for developing, maintaining certain aroma constituents in the
infant formula and an aroma or fragrance composition to be used to
increase the acceptance of a person or an object by the baby or new born.

22. A nutraceutical formulation according to claim 21 which comprises an
odorant selected from the group consisting of at least two, or at least
three, or at least four, or at least five, or at least six, or at least
seven or at least eight, or at least nine, or at least ten or at least
11, or at least 12, or at least 13, or at least 14, or least 15, or at
least 16 or at least 17, or at least 18, or at least 19, and at least 20
odorants as defined in claim 1.

24. A nutraceutical formulation according to claim 23, further comprising
an odorant selected from the group consisting of
5.alpha.-androst-16-en-3.alpha.-one, 4,16-androstadien-3-one, and
l,4androstadien-3,17-dion.

26. A nutraceutical formulation according to claim 21, wherein the
nutraceutical composition is in form of an infant formula or infant food.

27. A nutraceutical formulation according to claim 21, wherein the
nutraceutical composition is used for improving a baby's or newborn's
acceptance of a nutraceutical composition, especially infant formula.

33. A method according to claim 32 wherein said objects are selected from
the group consisting of breast comfort aide systems, pacifiers, sucking
devices, toys, pillows, clothing, incubators and medical equipment.

34. A method according to claim 32 wherein the scenting is applied in form
selected from a group consisting of a cream, ointment, lotion, emulsion,
oil, suspension, varnish, and solution.

40. The composition according to claim 39, wherein said composition is a
topical composition selected from the group consisting of a balm,
perfume, cream, ointment, lotion, body milk, emulsion, oil, suspension,
nail varnish and a solution.

41. The composition according to claim 39, wherein said composition is in
form selected from the group consisting of a balm, perfume, cream,
ointment, lotion, body milk, emulsion, oil, varnish, suspension, nail
varnish and a solution, for scenting an object.

42. A method for analysis of milk comprising, the use of sorptive
extraction from said milk.

Description:

BACKGROUND OF THE INVENTION

[0001]Breast-feeding is believed to exhibit a series of advantages for the
newborn compared to bottle-feeding. Human milk has been shown to impart
several nutritional and health benefits, for example promotion of the
infant's neuronal and intestinal development and fortification of the
immune system 1, 2, 3. Apart from these aspects, many studies point out
that human milk exhibits certain olfactory clues that foster the
newborns' ability to orient themselves versus the human milk source 4, 5,
6, and that might even play an important role in the development of
certain food preferences in later life 7, 8, 9, 10. Moreover, newborns
are able to distinguish between human milk and cow's or artificial milk,
they can separate between their own mother's Milk and that of other
women, and are even able to distinguish between different types of human
milk according to their individual temporal needs, that means between
e.g. colostrum and mature human milk 4, 6, 11, 12. Even more interesting
is that it was shown in these studies that babies prefer the odor
attributes of human milk compared to those of artificial infant formula
or milk products based on milk compositions of animal origin such as
cow's milk etc. To date, most studies in the area of milk research dealt
with the characterization of odor-active volatiles in processed animal
milks, predominantly UHT or pasteurized milks 13, 14. From these, the
conspecific odor compositions of the milks from for example cows, ewes,
etc. cannot be deduced as odorants might be degraded during the heating
and further processing steps, or might be formed as new and additional
odor constituents. Several effects forming artificial non-natural flavor
attributes in milk have been well-documented such as the sunlight-flavor
etc. 13, 14.

[0002]On the other hand, there are some studies dealing with the
identification and quantification of conspecific volatiles or odorants in
fresh or raw animal milk 14. However, most of these studies are based on
techniques such as gas chromatographic and mass spectrometric
methodologies that do not allow any weighting of a possible sensory
contribution of the respective compounds 15. Only a few studies targeted
at the identification of odor-active substances in animal milk such as
water buffalo and ewe's milk 16, 17, 18, 19. Among the most common
volatiles were aldehydes, ketones, esters, lactones and several diverse
substances such as terpenes and aromatic compounds.

[0003]Also, as for human milk, the influence of mother's diet on the milk
odor profile compositions is not yet understood. For cow's milk and ewe's
milk, there are a few studies characterizing the changes of milk odor
profiles with regard to specific feeding regimes, but in most cases just
on a sensory, not on a chemical-analytical basis 19, 20, 21.

[0004]While there is numerous evidence on the psychophysics level that
human milk displays a very characteristic and individual olfactory
composition, the molecular principles behind these phenomena remained
relatively unclear. This was predominantly due to the fact that, on the
one hand, sample size is very limited for comprehensive investigation of
the odor composition and of the odor-dynamics of human milk in relation
to single breastfeeding episodes. On the other hand, the commonly used
techniques for odor analysis of human milk such as solvent extraction
techniques or gas chromatographic analysis in coupling with mass
spectrometry or flame ionization detection were very limited with regard
to odor specificity.

[0005]This might be regarded as the main reason that only limited data on
the chemical characterization of odor-active compounds in human milk is
available to date 22. In this cited study, authors succeeded in
identifying a total of five odorants which were detectable in all four
analyzed breast milk samples. These compounds were the fatty-tallowy
smelling (E)non-2-enal, the mushroom-like smelling oct-1-en-3-one, the
caramel-like compounds 4-hydroxy-2,5-dimethyl-3(2H)-furanone and maltol,
and the milky smelling 2-nonanone. All of these compounds were also
detectable in formula milk. On the other hand, some additional compounds
were identified in formula milk: the buttery smelling butane-2,3-dione,
the cooked potato-like smelling 3-methylthio propanal (methional) and the
meat-like smelling 2-methyl-3-furanthiol. In conclusion, no human
milk-specific compound has been reported in this study. A second study
reported a series of volatiles in human milk, with 6 esters, 13 ketones,
6 fatty acids, 2 lactones, 24 aliphatic aldehydes, 9 alcohols, 18
hydrocarbons, and 6 miscellaneous compounds 23. Authors used an isolation
procedure based on simultaneous steam distillation-extraction under
reduced pressure using diethyl ether as the solvent with a distillation
temperature of 62 to 65° C. during two hours of extraction.
Detection and identification of the volatile compounds was accomplished
by means of gas chromatography combined with mass spectrometry based on
comparison of mass spectral data with library MS spectra. However, in the
cited study, the sensory properties and the sensory contribution or
impact of the identified compounds with regard to human milk was not
elucidated. Moreover, the list of detected volatiles comprises a series
of substances which are, to the specialist of the field of odor research,
highly unlikely to be odor-active contributors of human milk, for example
toluene, n-propylbenzene, 1,2,4-trimethylbenzene, m-, p- and
o-ethyltoluene, 1H-pyrrole, N-butyl-N-nitrosobutanamine,
N,N-dibutylformamide etc. Identification of such volatiles is more likely
to be produced either by artifacts, by drawbacks of the analytical
outline (identification by comparison with mass spectra library data
only) or by environmental contaminations of the human milk as it has been
described in different aspects 24, 25. In conclusion, it seems highly
improbable that these substances are natural human milk odor constituents
that are supposed to compose the human milk aroma attributes. This is
supported by the aspect that there are no common biochemical routes known
that would explain the occurrence of such substances in human milk as
endogenous compounds. In addition, it is not clear whether some of the
given volatiles have been generated due to the thermal treatment during
the analytical procedure as specified above, or have been destroyed.
Therefore, it is not possible to identify potential odor contributors for
human milk aroma among the identified volatiles presented in the cited
study, and to rate their possible impact and contribution to human milk
aroma.

[0006]So far the addition of natural odorants of human milk for improving
the acceptance of nutraceutical compositions has not been described.

[0007]In order to find an optimal infant formula, wherein the amounts and
composition of the odor-active substances are as dose as possible to
mother's milk, the present inventor has studied the odor composition of
fresh human milk by appropriate analytical tools without thermal
exposition, predominantly taking into account the aspect of
odor-activity. The analytical approach comprising gas
chromatographic-olfactometric characterization of the odor volatiles was
used that allowed the unambiguous identification of odor-active compounds
even in small-scale human milk samples.

[0008]This approach offers the possibility to analyze milk from individual
donors with minimal disturbance of the normal breast feeding procedure,
and without the need to pool samples from a number of donors, as it was
done in other studies. Also, the technique allows the analysis of
separate individual milk sample portions from within one feeding session.
That means the target was to develop an approach that allowed collection
of a small portion of milk right at the beginning of the feeding episode,
followed by one or more additional sampling events later on during the
same feeding episode.

[0009]To achieve this goal, a very versatile and sensitive extraction
technique for gaseous and liquid samples, the stir bar sorptive
extraction (SBSE) was used 26. In SBSE, a PDMS-coated stir bar is exposed
for a certain extraction time to a certain volume of sample either with
or without preliminary application of derivatization techniques. After
sorption of the analytes into the PDMS material, and removal of the
matrix system, the analytes can be easily recovered via solvent
extraction or thermo-desorption, and analyzed for example by means of
high resolution-gas chromatography or liquid chromatography in
combination with the respective detector systems. Compared to other
sorptive sample preparation techniques such as SPME, the SBSE has several
advantages such as convenient handling, high extraction capacity, very
low amounts of PDMS breakdown products and many more 27. Apart from
environmental investigations such as pesticide analysis and several
others 27, 28, 29, first applications of SBSE have been reported for the
direct analysis of e.g. benzoic acid or dicarboximide fungicides in foods
and beverages 30, 31, for the elucidation of biochemical pathways, and
for the analysis of odorous compounds in foods, mainly liquids 33, 34,
35. Combination with multidimensional gas chromatography using chiral
chromatography systems allowed the assignment of the stereochemistry of
aroma compounds in foods such as strawberries 36. In-vitro studies of
biological markers, drugs, their metabolites or other artificial
contaminants such as PCBs have been just recently performed on body
fluids such as sperm, blood and urine 37, 38. Recently, SBSE was used in
the context of a new in-vivo approach called BOSS (Buccal Odor Screening
System) 39, 40. It was successfully applied for the characterization of
volatile coffee and wine aroma "aftertaste" substances within the oral
cavity. A variation of the system was applied to monitor the development
of breath odor profiles after consumption of beer 43. The use in milk has
not been described. An adopted approach for characterization of human
milk odorants will be presented in the following.

[0010]Trace volatile and odorous substances that were characterized in
fresh human milk with regard to their molecular composition and their
sensory characteristics. The methodology was successfully applied for
identification of more than forty characteristic odorants in human milk.
The technique comprises a modified stir bar sorptive extraction system in
combination with two-dimensional gas chromatographic separation and
parallel mass spectrometric and olfactometric characterization of the
analytes. The present invention shows that the technique can be used both
for direct extractive sampling, but also for headspace analysis. Due to
its applicability for small sample volumes, even day-to-day physiological
variations in the profiles of volatile organic compounds in human milk
samples, but also fluctuations within e.g. one breastfeeding episode can
be monitored.

[0011]Accordingly, the object of the present invention is to provide
nutraceutical compositions that are more attractive for babies and
newborns.

SUMMARY OF THE INVENTION

[0012]The finding of the present invention is to select specific odorants
of human milk to use them in nutraceutical compositions.

[0013]The present invention relates to the use of odorants of the human
milk for improving the acceptance of nutraceutical compositions for
babies and newborns.

[0014]The preferred nutraceutical composition is infant formula. By
addition of the identified odorants or key odorants the acceptance of the
food product by the baby or newborn is enhanced.

[0015]Further the acceptance of objects that come into contact with babies
or newborns may be improved. Especially the acceptance of breast comfort
aid systems can be improved when flavored with the identified odorants or
mixtures thereof.

[0016]In addition the acceptance of other products such as pacifier,
pillows or toys may be improved.

[0017]A further aspect relates to the improvement of acceptance of
persons. Especially parents or caregivers being scented with the
identified odorants might endure less rejection by the babies or
newborns.

[0018]A further aspect relates to the general comfort of the baby. The use
of the identified odorants will help to pacify a baby.

[0019]Further the invention relates to the use of sorptive technique for
analyzing milk or infant formulas.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020]In the following the odorants of the invention are classified in the
following lists:

[0026]The present invention relates in one embodiment (1) to a
nutraceutical composition for newborns or babies comprising at one of the
odorants of LIST 1 or mixtures thereof. The composition may also comprise
at least two, or at least three, or at least four or at least five, or at
least six, or at least seven or at least eight, or at least nine, or at
least ten or at least 11, or at least 12, or at least 13, or at least 14,
or least 15, or at least 16 or at least 17, or least 18, or least 19, or
least 20 odorants of LIST 1.

[0027]Preferably in another embodiment (2) the composition, i.e. the
nutraceutical composition according to this invention, comprises at least
one of the odorants of LIST 2 or LIST 3 or mixtures thereof. The
compositions might comprise at least one additional odorant selected from
LIST 1. The composition may also comprise at least two, or at least
three, or at least four or at least five, or at least six, or at least
seven or at least eight, or at least nine, or at least ten or at least
11, or at least 12, or at least 13, or at least 14, or least 15, or at
least 16 or at least 17, or at least 18, or at least 19, or at least 20
additional odorants of LIST 1.

[0028]Preferably in a further embodiment (3) the composition, i.e. the
nutraceutical composition according to this invention, comprises at least
one compound of LIST 4.

[0029]The composition may also comprise at least two, or at least three,
or at least four or at least five, or at least six, or at least seven or
at least eight, or at least nine, or at least ten or at least 11, or at
least 12, or at least 13, or at least 14, or least 15, or at least 16 or
at least 17, or at least 18, or at least 19, or at least 20 additional
odorants of LIST 1.

[0030]In an especially preferred embodiment (4), said nutraceutical
composition comprises at least two, or at least three, or at least four
or at least five, or at least six, or at least seven or at least eight,
or at least nine, or at least ten or at least 11, or at least 12, or at
least 13, or at least 14, or least 15, or at least 16 or at least 17, or
at least 18, or at least 19, or at least 20 odorants of LIST 4. More
preferred the composition comprises at least 4 compounds of LIST 4.
Especially preferred, the nutraceutical composition comprises only
odorants of human milk as listed in List 4.

[0031]Additionally, in one of the above-mentioned embodiments (1) to (4),
compounds of LIST 5 may be added.

[0032]A preferred composition comprises at least one of the following
compounds: 5α-androst-16-en-3α-one,
δ4,16-androstadien-3-one, 1,4-androstadien-3,17-dion. The
composition may comprise at least one additional odorant selected from
LIST 1 or preferably LIST 4.

[0033]Especially, a preferred nutraceutical composition relates to infant
formula or infant food. Infant formula may be in powder or liquid form.

[0034]Further, the invention relates to a process for the manufacture of
nutraceutical compositions characterized by the addition of an odorants
as described for the nutraceutical composition. Especially, the invention
relates to manufacture of infant formula or infant food.

[0035]The odorant might be added as pure compounds or as mixtures with
other nutrients. For example in LIST 1 defined degradation products of
fatty acids might be added in combination with fatty acids.

[0036]The same applies for proteins, vitamins, or carbohydrates such as
oligosaccharides or lactose. In such a case the presence and the amount
of every above mentioned odorant might be determined during the
production of the nutraceutical composition.

[0037]Thus, the invention also relates to the determination of the
presence and quantification of at least one odorant of LIST 1 or
preferably LIST 2 or LIST 3 or LIST 4 in nutraceutical compositions for
babies.

[0038]If at least one of the above mentioned odorants was detected and
quantified, the concentration of the at least one odorants might be
adjusted to a concentration as indicated in table 2. The concentrations
relate to the composition ready to be administered to the baby.
Consequently, the concentrations of a concentrate have to be adjusted
accordingly. Typically the concentration of the odorant in a
powder-concentrate is 5-15 times, preferably about 10 times above the
final concentration.

[0039]Thus, the invention also relates to the determination and
quantification of at least one odorant of LIST 1 or preferably LIST 2 or
LIST 3 or LIST 4 in nutraceutical compositions for babies, whereby the
concentration of the odorants is adapted to give a nutraceutical
composition ready to be administered to babies that has the concentration
of the odorant as listed in table 2.

[0040]Further, the ratio between the at least one identified odorant and a
further odorant might be adjusted according to ratios derivable from the
concentrations indicated in table 2.

[0041]Thus, the invention relates to a method for improving the baby's or
newborn's acceptance of a nutraceutical composition, especially infant
formula, by using at least compound selected from LIST 1, formula. The
method also comprises the use of at least two, or at least three, or at
least four or at least five, or at least six, or at least seven or at
least eight, or at least nine, or at least ten or at least 11, or at
least 12, or at least 13, or at least 14, or least 15, or at least 16 or
at least 17, or least 18, or least 19, or least 20 odorants of LIST 1.

[0042]Preferably the method comprises the use of at least one of the
odorants of LIST 2 or LIST 3 or mixtures thereof. The method also
comprises the use of at least one additional odorant selected from LIST
1. The method also comprises the use of at least two, or at least three,
or at least four or at least five, or at least six, or at least seven or
at least eight, or at least nine, or at least ten or at least 11, or at
least 12, or at least 13, or at least 14, or least 15, or at least 16 or
at least 17, or at least 18, or at least 19, or at least 20 additional
odorants of LIST 1.

[0043]Preferably the method comprises the use of at least one compound of
LIST 4. Also at least two, or at least three, or at least four or at
least five, or at least six, or at least seven or at least eight, or at
least nine, or at least ten or at least 11, or at least 12, or at least
13, or at least 14, or least 15, or at least 16 or at least 17, or at
least 18, or at least 19, or at least 20 additional odorants of LIST 1
may be used.

[0044]Preferably at least two, or at least three, or at least four or at
least five, or at least six, or at least seven or at least eight, or at
least nine, or at least ten or at least 11, or at least 12, or at least
13, or at least 14, or least 15, or at least 16 or at least 17, or at
least 18, or at least 19, or at least 20 odorants of LIST 4 are used.
More preferred at least 4 compounds of LIST 4 are used.

[0045]Additionally, compounds of LIST 5 may be added.

[0046]A preferred method comprises the use of at least one of the
following compounds: 5α-androst-16-en-3α-one,
δ4,16-androstadien-3-one, 1,4-androstadien-3,17-dion, optionally in
addition of at least one odorant selected from LIST 1 or preferably LIST
4.

[0047]Further the invention relates to a method of improving the
acceptance of objects that come into contact with the baby or newborn by
scenting these with an odorant selected from LIST 1 or mixtures thereof.
The method also comprises the use of at least two, or at least three, or
at least four or at least five, or at least six, or at least seven or at
least eight, or at least nine, or at least ten or at least 11, or at
least 12, or at least 13, or at least 14, or least 15, or at least 16 or
at least 17, or least 18, or least 19, or least 20 odorants of LIST 1.

[0048]Preferably the method comprises the use of at least one of the
odorants of LIST 2 or LIST 3 or mixtures thereof. The method also
comprises the use of at least one additional odorant selected from LIST
1. The method also comprises the use of at least two, or at least three,
or at least four or at least five, or at least six, or at least seven or
at least eight, or at least nine, or at least ten or at least 11, or at
least 12, or at least 13, or at least 14, or least 15, or at least 16 or
at least 17, or at least 18, or at least 19, or at least 20 additional
odorants of LIST 1.

[0049]Preferably the method comprises the use of at least one compound of
LIST 4. Also at least two, or at least three, or at least four or at
least five, or at least six, or at least seven or at least eight, or at
least nine, or at least ten or at least 11, or at least 12, or at least
13, or at least 14, or least 15, or at least 16 or at least 17, or at
least 18, or at least 19, or at least 20 additional odorants of LIST 1
may be used.

[0050]Preferably at least two, or at least three, or at least four or at
least five, or at least six, or at least seven or at least eight, or at
least nine, or at least ten or at least 11, or at least 12, or at least
13, or at least 14, or least 15, or at least 16 or at least 17, or at
least 18, or at least 19, or at least 20 odorants of LIST 4 are used.
More preferred at least 4 compounds of LIST 4 are used.

[0051]Additionally, compounds of LISTS may be added.

[0052]A preferred method comprises the use of at least one of the
following compounds: 5α-androst-16-en-3α-one,
δ4,16-androstadien-3-one, 1,4-androstadien-3,17-dion, optionally in
addition of at least one odorant selected from LIST 1 or preferably LIST
4.

[0054]The scenting might be added during the production process or after
manufacture. The object may be impregnated or a composition may be
applied which comprises at least one of the above mentioned odorants.
Such a composition may be selected from cream, ointment, lotion,
emulsion, oil, suspension, varnish, or solution.

[0055]Thus invention relates to a cream, ointment, lotion, emulsion, oil,
suspension, varnish, or solution, comprising the odorants as described
above.

[0056]Further the invention relates to a method of improving the
acceptance of a person by babies or newborn or to a method of pacifying a
baby by using a composition with an odorant selected from LIST 1 or
mixtures thereof. The method also comprises the use of at least two, or
at least three, or at least four or at least five, or at least six, or at
least seven or at least eight, or at least nine, or at least ten or at
least 11, or at least 12, or at least 13, or at least 14, or least 15, or
at least 16 or at least 17, or least 18, or least 19, or least 20
odorants of LIST 1.

[0057]Preferably the method comprises the use of at least one of the
odorants of LIST 2 or LIST 3 or mixtures thereof. The method also
comprises the use of at least one additional odorant selected from LIST
1. The method also comprises the use of at least two, or at least three,
or at least four or at least five, or at least six, or at least seven or
at least eight, or at least nine, or at least ten or at least 11, or at
least 12, or at least 13, or at least 14, or least 15, or at least 16 or
at least 17, or at least 18, or at least 19, or at least 20 additional
odorants of LIST 1.

[0058]Preferably the method comprises the use of at least one compound of
LIST 4. Also at least two, or at least three, or at least four or at
least five, or at least six, or at least seven or at least eight, or at
least nine, or at least ten or at least 11, or at least 12, or at least
13, or at least 14, or least 15, or at least 16 or at least 17, or at
least 18, or at least 19, or at least 20 additional odorants of LIST 1
may be used.

[0059]Preferably at least two, or at least three, or at least four or at
least five, or at least six, or at least seven or at least eight, or at
least nine, or at least ten or at least 11, or at least 12, or at least
13, or at least 14, or least 15, or at least 16 or at least 17, or at
least 18, or at least 19, or at least 20 odorants of LIST 4 are used.
More preferred at least 4 compounds of LIST 4 are used.

[0060]Additionally, compounds of LISTS may be added.

[0061]A preferred method comprises the use of at least one of the
following compounds: 5α-androst-16-en-3α-one,
δ4,16-androstadien-3-one, 1,4-androstadien-3,17-dion, optionally in
addition of at least one odorant selected from LIST 1 or preferably LIST
4.

[0062]The composition might be applied topically to the skin or clothing
of the person. An area where the composition is to be applied may be the
breast, especially woman's breast. The composition may be applied in form
of a balm, perfume, cream, ointment, lotion, body milk, emulsion, oil,
suspension, nail varnish or solution.

[0064]Persons that may benefit from the use of above described odorants
include parents, caregivers and medical staff.

[0065]Moreover the invention relates to a method of analyzing milk or
infant formulas characterized by the use of sorptive extraction. The
sorptive extraction may be combined with gas chromatographic separation.
Preferably the sorptive device is introduced into the liquid. Preferably
the absorbent/adsorbent material is polydimethylsiloxane (PDMS). The
preferred adsorbent device is a stir bar, a sheet, or beads or powder.
The beads or the powder may be encapsulated in a capsule with pores or
any other kind of openings. Preferably the device is a stir bar.
Preferably the method is used for the analysis of odorants.

[0066]Whole human breast milk may be excluded from all compositions
described herein. Also compositions comprising whole human breast milk
and additional components may be excluded. By the same token whole animal
milk, especially cow, ewe, goat, horse, donkey, lama or camel milk may be
excluded. Also compositions which contain whole animal milk where
additives have been added may be excluded. The term animal milk should
include raw, heated or UHT milk.

[0068]Nutraceutical compositions for babies and newborn include any type
of nutraceutical composition adapted for babies or newborn. Preferred are
infant formula and infant food, especially infant formula. The infant
formula may be in powder or liquid or semi-liquid form. The infant
formula may be based on recommendations by the American Academy of
Pediatrics Committee on Nutrition. The following should be included at
least:

[0070]In addition, biotin, choline, inositol should be included. Normally
they are based on cow milk or soy milk. Infant formula may also be based
on milk from other animals such as camel, ewe or goat.

[0071]The infant formula will be adapted for premature babies, for newborn
babies up to one month, for babies up to three months, for babies up to
four months, for babies up to five months, for babies up to six months,
or babies up to 1 year. The infant formula may be designed for babies
from birth to four months. The infant formula nay be adapted for baby
from the forth month or from the eighth month. Preferably the infant
formula is designed for premature babies, for babies up to one month, for
babies up to four months, or for babies up to six months.

[0072]The term nutraceutical composition comprises also concentrates which
have to be diluted before administration to the baby.

[0073]The term newborn or baby relates to any baby including premature
babies, babies up to four months, up to six months or babies up to one
year. Preferably the term relates to premature babies or babies up to six
months.

[0075]Ten samples of human milk were collected from different donors and
immediately stored for a maximum of two days prior to analysis at
-18° C. Panelists were non-pregnant volunteers (non-smokers,
Germans of Caucasian ethnicity) in the age range 28-35 (mean age 32),
exhibiting no known illnesses at the time of examination. Breast milk
production was normal and no breast infection was detected prior to milk
sampling. Sampling took place in the lactation period from 12 to 20 weeks
postpartum. 20 mL samples were taken during the day, about 1-2 hours
after a light meal that was relatively weak in odor (water, plain bread
with little amounts of butter and cottage cheese), right before a regular
breast feeding episode. Prior to sample collection and analysis, written
consent has been obtained from all participants providing human milk
samples after full explanation of the purpose and nature of the study.

PDMS-coated Stir Bars

[0076]For the experiments, commercially available Twister®-SBSE bars
(20 mm length, 0.5 mm POMS coating thickness and ˜50 μL of total
POMS volume according to the suppliers specifications; Gerstel GmbH,
Muhlheim a/d Ruhr, Germany) were used. Prior to analysis, the bars were
subjected to a conditioning procedure according to the suppliers
recommendations: the stir bars were first soaked in 100% acetonitrile for
at least two days, then conditioned at 300° C. for 4 h.

[0077]Each SBSE bar was first screened for odorants ("background", see
"Results and Discussion") and then directly used for analysis. Each stir
bar was used for just one single experiment, then reconditioned and
screened for background again. Each experiment was performed with at
least three different SBSE bars to avoid SBSE bar variations.

Direct StirBar Sorptive Extraction of Human Milk Samples

[0078]Five mL of each human milk sample were pipetted separately into 10
mL closed glass vessels and tightly sealed with a lid (cf. FIG. 1). Glass
vessels had been thoroughly cleaned prior to analysis and
heat-conditioned at 110° C. to avoid any odorous contamination. A
SBSE bar was immediately placed in the respective sample, stirred for 1
h, removed with tweezers, dipped into deodorized water, briefly dried
with lint-free tissue and immediately placed into the thermo-desorption
unit.

Headspace Sampling

[0079]Five mL of each human milk sample were pipetted separately into 10
mL closed glass vessels, affixed with inserts for headspace exposition of
the PDMS stir bar, and tightly sealed with a lid (cf. FIG. 1). Glass
vessels had been thoroughly cleaned prior to analysis and
heat-conditioned at 110° C. to avoid any odorous contamination.
Equilibration of the SBSE bar within the headspace insert above the
stirred sample was conducted for 1 h. Then, the SBSE bar was removed with
tweezers, dipped into deodorized water, briefly dried with lint-free
tissue and immediately placed into the thermo-desorption unit.

SBSE Thermo-Desorptive Sample Application

[0080]Thermo-desorption of the samples was performed by means of a TDS-2
thermo-desorption system (Gerstel GmbH) in combination with a CIS-4 PTV
injector (Gerstel GmbH) for cryofocussing the analytes prior to transfer
onto the analytical column. The following sampling parameters were used:
Splitless thermal desorption was performed by programming the TDS-2 from
40° C. to 240° C. (5 min) at a rate of 60° C.
Cryofocussing was performed with liquid nitrogen at -100° C.
Injection was performed with a ramp of 12° C./s from -100°
C. to 240° C. (5 min). The gas chromatographic conditions are
given below.

High Resolution Gas Chromatography-Olfactometry

[0081]Application of the samples was performed as described above (SBSE
Thermo-Desorptive Sample Application). The odorants were screened in
parallel by three panelists by sniffing the effluent after gas
chromatographic separation. Sniffing analysis was repeated three times by
each panelist. All detected odorants were identified by comparison with
reference substances on the basis of the following criteria: retention
index (RI) on two stationary phases of different polarity (DB-FFAP,
DB-5), mass spectra obtained by MS (EI) and MS (CI); and odor quality as
well as odor intensity perceived at the sniffing-port. Only if the odor
quality and intensity of the reference agrees with that detected via
GC-O, identification can be regarded as "positive".

[0083]The odorants were analyzed by two-dimensional gas chromatography
(TD-HRGC) as described above. MS analyses were performed in parallels
with the sniffing analysis on the main column system with an ITD-8OO
(Fisons Instruments, Mainz-Kastel, Germany) running in the CI-mode with
methanol as the reagent gas. The following fused silica columns were
used: DB-FFAP (30 m×0.32 mm i.d., 0.25 μm FD, J & W Scientific,
Folsom, USA) in combination with DB-5 (SE-54; 30 m×0.32 mm i.d.,
0.25 μm FD, J & W Scientific, Folsom, USA). The gas chromatographic
and mass spectrometric conditions were the same as described previously.

[0084]When analyzing the headspace above the human milk samples by means
of HRGC-olfactometry of the adsorbed substances, a total of 22
odor-active compounds was detected.

[0085]Twenty-one of these were identified based on the criteria given in
the experimental section (cf. Table 2). Among the detected compounds were
a series of fatty smelling compounds, (E)-oct-2-enal, (Z)-non-2-enal,
(E)-non-2-enal, (E,E)-nona-2,4-dienal, grassy, leaf-like and citrussy
substances (hexanal, octanal), and several coconut-like smelling
compounds of the lactone group, that is γ-nonalactone,
γ-decalactone, and δ-decalactone. Acidic and sweaty
impressions were due to acetic acid and butanoic acid. Apart from that,
several sweet substances were detectable, such as the honey-like smelling
phenylacetic acid, as well as 4-hydroxy-2,5-dimethyl-3(2H)-furanone
(caramel-like) and vanillin (vanilla-like). Metallic, mushroom- and
geranium leaf-like substances were hex-1-en-3-one, oct-1-en-3-one,
(Z)-octa-1,5-dien-3-one and tr-(4,5)-epoxy-(E)-2-decenal and an
unidentified geranium leaf-like smelling odorant.

[0086]Specific malty, buttery and cooked potato-like impressions were
elicited by methylpropanal, diacetyl, and methional, respectively. With
the exception of the unknown geranium leaf-like compound, all odorants
were detectable in all milk samples from different donors (Table 1).

[0087]The chemical structures of these odorants are shown in FIGS. 3 and
4.

[0088]HRGC-olfactometric analysis of the adsorbed volatiles from direct
extraction of human milk led to the identification of the same compounds
discussed above (cf. Table 1). Apart from that, 25 additional compounds
were detectable by means of HRGC-O. Only seven of these were detectable
in all human milk samples from all donors: the peach-like smelling
(Z)-6-γ-dodecenolactone, the fatty smelling (E,E)-deca-2,4-dienal,
and the citrussy-soapy smelling compounds nonanal and decanal.
Interestingly, the other three of these seven odorants elicited for
adults relatively objectionable, animalic odors: the goat-like smelling
4-ethyloctanoic acid, an unidentified musty smelling odorant, as well as
the urine-like smelling 5α-androst-16-en-3α-one.

[0089]Additional compounds were the faeces-like smelling 3-methylindole,
the intensely sweaty 2- and 3-methylbutanoic acids and pentanoic acid,
and an additional unidentified goat-like compound. These odorants were,
however, not sensorically detectable in all milk samples. The remaining
compounds were also not detectable in all milk samples, but only a few of
them. These were the fatty and cucumber-like smelling odorants
(E,E)-octa-2,4-dienal and (E,Z)-nona-2,6-dienal, and the mushroom-like
smelling non-1-en-3-one. Other for adults relatively pleasant odor notes
originated from the flowery compounds linalool and β-ionone, the
sweet smelling 2-aminoacetophenone and δ-dodecalactone, and two
honey-like odorants (phenylacetaldehyde and 2-phenylethanol).
Characteristic bellpepper-like, phenolic and savory-like impressions were
elicited by 3-isobutyl-2-methoxypyrazine, 2-methoxyphenol, and sotolon,
while one metallic smelling compound remained unidentified.

[0090]In the present invention, a broad variety of odor-active substances
was identified in human milk samples. It was shown that not only a large
number of compounds can be found, but that these odorants are
structurally very diverse, and exhibit an array of different olfactory
impressions. It was also shown that most of these substances were
detectable either by means of headspace analysis or by direct SBS
extraction of the milk samples. For this reason these compounds are
characteristic constituents of human milk.

[0091]Comparing the results presented here with those of Bingham et al.
(Bingham P M, Stevens-Tuttle D, Lavin E, Acree T. Arch. Pediatr. Adolesc.
Med. 2003, 157: 1031) who analyzed four breast milk samples, it can be
seen that the fatty smelling (E)-non-2-enal, the mushroom-like smelling
oct-1-en-3-one and the caramel-like smelling
4-hydroxy-2,5-dimethyl-3(2H)-furanone were in both studies among those
compounds which were detectable in all milk samples. However, diacetyl
and methional which were only found in formula milk in the previous
study, were identified as human milk odorants in the present application,
together with more than 30 additional odorants which had not been
reported by Bingham et al.

[0092]More than 50% of these odorants were even detectable just in the
headspace of the human milk samples, without the need for a direct milk
extraction methodology. This displays the high sensitivity and
selectivity of the applied analytical procedure. On the other hand, those
compounds which were additionally detected by direct SBS extraction of
the milk samples were odorants with relatively high molecular weight,
such as 5α-androst-16-en-3α-one or
(Z)-6-γ-dodecenolactone. Often they were homologues with higher
molecular weights of related substances already detected by headspace
analysis. For example, hexanal and octanal were identified by means of
headspace analysis while nonanal and decanal were additionally found by
the direct extraction procedure. This mirrors discrimination effects of
the headspace technique due to volatility aspects, and highlights the
need to use the direct extraction technique as complementary tool.

[0093]The detection of the steroid compound
5α-androst-16-en-3α-one as sensorically active constituent
using the presented SBSE procedure is noteworthy. On the other hand,
maltol and 2-nonanone were not detected in our study by means of HGRC-O
after SBSE enrichment. It is difficult to speculate about the reasons for
these differences as details on the exact experimental procedure have not
been provided in the previous study (Bingham P M, Stevens-Tuttle D, Lavin
E, Acree T. Arch. Pediatr. Adolesc. Med. 2003, 157: 1031). While it has
been specified that analysis and identification had been performed by
means of gas chromatography-olfactometry, and had also been based on the
respective odorant retention indices, no details have been given on the
mode of odorant enrichment or recovery. In this context, it needs to be
stated that only those compounds were taken into consideration within the
present invention which were detectable by means of HRGC-O, but not
HRGC-FID or HRGC-MS only. This has obviously been done accordingly in the
study by Bingham et al. Therefore, the detection differences are very
likely due to the fact that another sample work-up procedure has been
used, such as solvent extraction and/or a distillation approach.

[0094]Generally, it has to be noted that maltol and 2-nonanone are not
very potent odorants. Comparison with previous results on SBSE
application in the determination of trace aroma compounds in
physiological context implies that this approach is a highly sensitive,
and a useful complementary tool for identification of odorants in
small-scale samples. This is affirmed by the present study with the
first-time characterization of large number of human milk odorants. This
shows that the applied technique can be used as a screening system for
potent odorants in small-scale human milk samples. For selective and
sensitive quantitation the presented technique can be used as basis for
e.g. stable isotope dilution assays as described elsewhere.